Antiserum



. Patented June. 17,1941

v UNITED STATES PATENT OFFICE 2,246,355 I AN'rIsEaUil Tillman D. Gerlough, Highland Park, N. .L, assigner to E. B. Squibb & Sons, New York, N. 'Y., acorporation of New York No Drawing; Application March 5, 1938,

Serial No. 194,082

i Claims. (or 167-78) This invention relates to, and has for its object the provision of, refined and concentrated type-specific antipneumococcic rabbit serum and a method of preparing it.

The efficacy of type-specific antipneumococcic resulting mixture about one-third saturated serum 1nlobar-pneumonia therapy is well recogwith the salt, filtering, adding to the filtrate a mzed in medical practice. Such antiserum has sufllcient quantity of the Saturated salt solution heretofore been prepared from the serum of to make the mixture about one-half saturated horses highly immunized against a specific type with the salt, separating the precipitated antiof pneumococcus; but Horsfall, Goodner, Macbody-containing protein, and removing the am- Leod, and Harris have recently pointed out monium sulfate therefrom. (J. A. M. A. 10821483, May 1, 1937) certain ad- Preferably, the saturated ammonium sulfate, vantages, both theoretical and practical, which solution used in the practice of this invention is antipneumococcic serum prepared from the rabprepared by dissolving ammonium sulfate (at 1 bit possesses over that from the horse. Of these, 25 C.) in distilled water (free of iron and other the most important from the therapeutic point, heavy metals), adding concentrated ammonia of view are: (l) the smaller size of the antibody water until the solution has a distinctodor of molecule in rabbit serum and the consequent ammonia, and filtering through hardened filter deeper penetration of the antibody into the inpaper, Preferably also, the distilled water used fected tissues; and (2) the greater titer of antishould be substantially pyrogen-free, and the rebody units obtained in rabbit serum. The intraagents used should be highly pure. venous administration of native (i. e., untreated) The following example is illustrative of the antipneumococcic rabbit serum, however, results invention: 34.9 liters of serum, obtained from in a high-incidence of chills. rabbits immunized with virulent pneumococcus lath practice of this invention, the serum of type I and containing about 2500 Felton mouse rabbits immunized to a high titer with virulent units per cc., is preserved with 0.02% of sodium pneumococci (e. g. type I) is subjected to a speethyl merourithiosalicylate and stored at a temcial refining and concentrating method to properature of 0-5 C. for 2 to 6 weeks; the preduce a highly-concentrated type-specific anticipitated fatty and proteinaceous material is filpneumococcic serum which has a relatively low tered oil, the clarified serum is diluted with an content of inactive protein and is substantially equal volume of distilled water free of iron and free of chill-producing substances (i. e., is charother heavy metals, and heated at 56 C. for flclferized by an extremely low incidence of about a half hour (any fibrin present, as when chills) Since the eflicacy of antipneumococcic plasma is usedpbeing thus coagulated); the re serum is dependent on the early administration suiting precipitate is removed by filtration of large amounts of antibody, the increased antithrough paper pulp, and sufficient saturated ambody-concentration made available by the remonium sulfate solution is added to make the fined and concentrated antipneumococcic rabbit resulting mixture about 30% saturated with amserum of this invention gives rise to an impormonium sulfate; after standing overnight at tant therapeutic advantage. Another feature as 40 0-10 C., the mixture is filtered through hardto which the antiserum ,of. this invention obviened filter paper; to the filtrate is added suffiously enjoys significant-superiority is the virtual cient saturated ammonium sulfate solution to elimination therefrom of the materials whose make the mixture about 50% saturated with ampresence brings about the distressing reaction 'monium sulfate, the precipitated antibody-conof chills. taining protein is filtered off, and the m o The procedureof this invention essentially comprises adding a preservative (e. 8., phenol,

cresol, or sodium, ethyl mercurithiosalicylate) to a native antipneumococcic serum (which term, asherein employed, includes plasma), aging the serum at a low temperature, removing .the precipitated fatty and proteinaceous material (as by filtering through paper pulp), diluting the soclarified serum with distilled water, heating the any fibrin present), removing the precipitate,

adding to the serum a sufficient quantity of a saturated solution of a highly-soluble neutral salt (preferably ammonium sulfate) to make the sulfate removed therefrom by dialysis. There is obtained 8 liters of refined and concentrated The invention may be variously otherwise em- 1 antipneumococcic serum type I, having a potency of about 10,000 Felton mouse units per cc.

bodied within the scope of the appended claims. I claim: i

1. The process of refining and concentrating type-specific antipneumococcic rabbit serum diluted serum to about 54-58 C. (to coagulate which comprises adding apreservative, aging the steps or diluting the serum with distilled water,

serum at a low temperature, removing the precipitate, diluting the so-clarifled serum with distilled water, heating to about 54-58 C... removing the precipitate, adding to the serum a sufiicient quantity of a saturated solution of ammonium sulfate to make theresulting mixture about one-third saturated with'the salt, filtering, adding tothe filtrate a sufiicient quantity or the saturated salt solution to make the mixture about one-halt saturated with the salt, separating the precipitated antibody-containing protein, and removing the salt therefrom.

2. The process of refining and concentrating antipneumo'coccic rabbit serum type I which comprises adding a preservative, aging the serum at a low temperature, removing the precipitate,

, diluting the so-ciarified serum with distilled water, heating to about iii-58 C., removing the precipitate, adding to the serum a suiiicient quantity or a saturated solution of ammonium sulfate to make the resulting mixture about one-third saturated with the salt, filtering, adding to the filtrate a 'sufiicient quantity or the saturated salt solution to make the mixture about one-half saturated with the salt, separating the precipitated antibody-containing protein, and removing the salt therefrom.

3. In the process of refining and concentrating p -specific antipneumococcic rabbit serum, the

removing the precipitate, adding to such serum a sufllcient quantity 01 a saturated solution of ammonium to make, the resulting mixture about one-third saturated with the salt, filtering, adding to the filtrate a sufllcient quantity of the saturated salt solution to make the mixture about one-half saturated with the salt, separating the precipitated antibody-containing protein, and removing the salt therefrom.

4. The process of refining and concentrating type-specific antipneumococcic rabbit serumwhich comprises adding 0.02% of sodium ethyl mercurithiosalicylate, storing the serum at a temperature oi 04 C. for 2 to 6 weeks, filtering, diluting the filtrate with an equal volume 0! distilled water, heating at 56 C, for about a half hour, filtering, adding suiiicient saturated ammonium sulfate solution to make the resulting mixture about saturated with ammonium sulfate, allowing the mixture to stand at 010 C., filtering, adding to the filtrate sufilcient saturated ammonium sulfate solution to make the mixture about saturated with ammonium sulfate, filtering, and removing the ammonium sulfate from the precipitate by dialysis.

, TmMAN D. GERLOUGH.

CERTIFICATE OF'CQRBECTIOR. Patent, NO. I I j 17- TILIMAN n. GERLOUGH. I

It is hereeycertified thalt error eppears 'in the printer; specification of the above mnpbered patent requiring cforrection as follows: Page 2, seeend column, line 1;, elaim 5, after theydord "Ammonium" insert- -sulfateand that the said Letters Patei'zt should be read with this cbrrectiontherein that the same inay conform to the reqord of the case in the Petent Office.

Signed and sealed. this 2 9th m of January, A. 15'. 19M.

Lesl ie Frazer (sea l) I First Assistant Cmmnissioner of Patenta 

